PROJECT SUMMARY/ABSTRACT Background and long-term objectives: This proposed research will advance our understanding of how the lung microbiome contributes to the pathogenesis and perpetuation of oxygen-induced lung injury. Inhaled oxygen is among our most commonly administered therapies. Yet hyperoxia - elevated inspired oxygen - causes lethal lung injury in animals, and in humans is associated with increased mortality and development of the acute respiratory distress syndrome. We have recently discovered that hyperoxia acutely alters lung microbiota. This oxygen-induced dysbiosis is strongly and temporally correlated with alveolar inflammation. We have discovered that germ-free mice - experimental mice devoid of microbiota - are protected from oxygen- induced lung injury, an observation that cannot be explained via our conventional model of oxygen-induced lung injury. Conversely, lung injury alters lung microbiota by changing bacterial growth conditions within the lung microenvironment. We have discovered that germ-free mice are protected from non-resolving lung injury (bleomycin), indicating that the microbiome is necessary for perpetuation of lung injury. The discovery of the lung microbiome has thus broadened our model of pathogenesis. The mechanisms by which lung microbiota mediate oxygen-induced lung injury, and are in turn altered by lung injury, are undetermined. The central hypothesis of this proposal is that specific bacteria within the lung ecosystem propel alveolar inflammation in oxygen-induced lung injury, and these bacteria are enriched within the lung microbiome both by hyperoxia itself and by the altered ecology of injured lungs. The rationale is that these discoveries will facilitate the development of therapies for the prevention and treatment of oxygen-related human lung disease. Specific Aim 1: To determine the microbial and molecular pathways by which oxygen therapy alters lung microbiota, mediating host inflammation and injury. We will accomplish this Aim by integrating complementary experimental approaches: in vivo heterogeneity analysis of host-microbiome interactions in mice; in vivo germ-free, gnotobiotic, and antibiotic-treated hyperoxia modeling in mice; data science interrogation of observational human data using a validated machine-learning algorithm. Specific Aim 2: To determine the molecular pathways by which oxygen-induced host inflammation and injury alter lung microbiota, perpetuating respiratory dysbiosis and lung injury. We will accomplish this Aim by integrating complementary experimental approaches: a novel ex vivo culture assay that identifies host- derived mediators of bacterial growth; in vivo augmentation and inhibition of the host response in hyperoxia. This translational research approach will determine 1) the key members of the lung microbiome that mediate oxygen-induced lung injury, 2) the pathways by which these bacteria promote alveolar inflammation, and 3) the ecologic factors within the injured lung environment that promote their growth.